Cysteine composition and injection

ABSTRACT

Cysteine compositions comprising less than about 400 μg/L of aluminum. For example, solutions of cysteine comprising a pharmaceutically acceptable solvent, cysteine, and less than about 145 μg/L of aluminum, wherein the solution is devoid of visible particulate matter. Cysteine compositions described herein may be suitable for injection. For example, disclosed cysteine solutions may be provided intravenously to meet amino acid nutritional requirement in individuals receiving total parenteral nutrition. Also provided are processes for preparing cysteine compositions, and methods for providing cysteine to individuals in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/373,255, filed Apr. 2, 2019, which is a continuation of and claimspriority to U.S. patent application Ser. No. 16/355,028, filed Mar. 15,2019.

FIELD

The present disclosure relates to cysteine compositions having less thanabout 400 μg/L of aluminum and/or exhibiting extended stability.

BACKGROUND

Cysteine is considered a semi-essential amino acid for newborn infantsand is routinely added to neonatal total parenteral nutrition (TPN)formulations. However, the stability of cysteine is low in solution,e.g., it can oxidize to form cystine, a disulfide dimer, which has alower solubility than cysteine. Most crystalline amino acid solutionshave pH levels of about 5-7, which would favor the conversion ofcysteine to cystine. For these reasons, cysteine has typically beenformulated as a separate parenteral nutrition additive and tends to bemixed with other TPN components just prior to use. Thus, preparations ofcysteine with improved stability would be beneficial.

Aluminum is a contaminant commonly found in parenteral nutritionadditive solutions. Research indicates that patients with impairedkidney function, including neonates, who receive parenteral levels ofaluminum at greater than 4 to 5 μg per kg per day accumulate aluminum atlevels associated with central nervous system and bone toxicity. In anattempt to limit the risk of aluminum toxicity, the U.S. Food and DrugAdministration (FDA) has rules limiting the total aluminum content inTPN formulations. Since some preparations of cysteine can contain up to5000 μg/L of aluminum, the total daily aluminum limit may be exceeded.Thus, preparations of cysteine with reduced levels of aluminum wouldalso be beneficial.

SUMMARY

Among the various aspects of the present disclosure is the provision ofsolutions of cysteine, each of which includes a pharmaceuticallyacceptable solvent (e.g., water), cysteine (e.g., cysteinehydrochloride), and less than about 400 μg/L of aluminum. In someembodiments, the aluminum content may be substantially lower than 400μg/L; for instance, the aluminum content of some embodiments may be lessthan about 145 μg/L, less than about 100 μg/L, less than about 30 μg/L,or even less than about 10 μg/L. In some embodiments, the aluminumcontent may range from about 0 μg/L to about 400 μg/L or from about 3μg/L to about 145 μg/L. The aluminum content of some solutions describedherein may be characterized with respect to specific storage conditions.For instance, the aluminum content of some embodiments may be less thanabout 400 μg/L when the solution is stored in a silica-lined vial i) for6 months at 40° C. and 75% relative humidity or ii) for 18 months at 25°C. and 60% relative humidity.

Some of the above-described solutions exhibit relatively low pHs and/orinclude low levels of dissolved oxygen content. For instance, somesolutions may have a pH from about 1.0 to about 2.5, a pH from about 1.0to about 1.5, or a pH from about 1.0 to about 1.3. As another example,some solutions may have a dissolved oxygen content of less than 2 mg/L.

The solutions of cysteine set forth above may be devoid of visibleparticulate matter and/or precipitated particles of cystine. Forinstance, a solution may be devoid of visible particulate matter whenstored for 18 months at 25° C. and 60% relative humidity. As anotherexample, a solution may be devoid of visible particulate matter whenstored for 6 months at 40° C. and 75% relative humidity.

Some embodiments of the solutions set forth above may be characterizedas being sterile and/or formulated for injection (e.g., intravenousinjection).

All of the features described above may exist individually or in anycombination. For instance, in some embodiments, the solution may becharacterized as having 50 mg/mL of cysteine hydrochloride monohydrate,less than 145 μg/L of aluminum, and a pH from about 1.0 to about 2.5, aswell as being devoid of visible particulate matter.

Another aspect of the present disclosure provides a silica-lined vialhaving a solution of cysteine (such as any of those described above)disposed therein. The solution in the vial may be overlaid with a layerof nitrogen. The vial may be sealed in any appropriate manner (e.g.,with a cap) and may include a pierceable septum or the like to enablethe solution to be drawn therefrom for administration (e.g., intravenousadministration) to individuals. In certain iterations, the solution inthe vial comprises 50 mg of cysteine hydrochloride monohydrate per mL ofwater for injection adjusted to pH 1.0 to 2.5. In some iterations, thevial comprises 10 mL of the solution, which may or may not be requiredto be diluted prior to administration to an individual in need thereof.

Yet another aspect of the present disclosure encompasses a totalparenteral nutrition formulation that includes a solution of cysteinedescribed herein.

Still another aspect of the present disclosure provides a solution ofcysteine having a cysteine monomer content of at least 99% by weight anda cystine dimer content of less than 1% by weight. The dissolved oxygencontent of the solution may be less than about 2 mg/L, and the pH of thesolution may be from about 1.0 to about 1.5. In some embodiments, thecystine dimer content of the solution may be less than about 0.5% byweight. The solution of this aspect is preferably devoid of precipitatedparticles of cystine dimer. In some embodiments of this aspect, thesolution may have an aluminum content of less than about 400 μg/L orless than about 145 μg/L.

Still another aspect of the present disclosure provides processes forpreparing cysteine having i) an aluminum content of less than about 400μg/L (e.g., the aluminum content of any of the solutions of cysteinedescribed above) and/or ii) a cystine dimer content of less than about1% (or even less than about 0.05%) by weight. In this process, cysteinepowder is added to a volume of a pharmaceutically acceptable solvent(e.g., water) to form a mixture. That mixture is mixed (e.g., stirred,agitated, or the like) to form a solution. The solution is then filteredto form a filtered solution, which can be dispensed into an appropriatecontainer (e.g., a vial, such as a glass vial that may have an interiorlining of silica). This process is preferably (but not necessarily)conducted under a nitrogen atmosphere.

In some embodiments of the above-described processes for preparingcysteine, the pharmaceutically acceptable solvent may be acidified(e.g., by addition of ⅛th molar equivalent of hydrochloric acid). Thisacidification typically (but not necessarily) would occur prior toadding the cysteine powder to the pharmaceutically acceptable solvent.As a result of this acidification, the pharmaceutically acceptablesolvent preferably exhibits a pH well below 5. For instance, in someembodiments, the resulting pH of the pharmaceutically acceptable solventmay be between about 1.0 and about 2.5 (e.g., a pH of from about 1.0 toabout 1.3).

Still with respect to the above-described processes for preparingcysteine, the pharmaceutically acceptable solvent may be degassed (e.g.,by sparging same with nitrogen). In certain iterations, the dissolvedoxygen content of the pharmaceutically acceptable solvent may be lessthan about 2 mg/L.

In some embodiments of the above-described processes for preparingcysteine, the filtered solution in the container may be overlaid with alayer of nitrogen, and the container may be sealed. In such embodiments,the filtered solution in the sealed container is preferably devoid ofvisible particulate matter. In some embodiments, the filtered solutionin the sealed container may be characterized as being devoid of visibleparticulate matter i) after storage for 6 months at 40° C. and 75%relative humidity or ii) after storage for 18 months at 25° C. and 60%relative humidity.

Another aspect of the present disclosure encompasses a method forproviding cysteine to a patient (e.g., neonate) in need of receivingtotal parenteral nutrition. In this method, the patient is administereda solution of cysteine described herein. The solution may beadministered in any appropriate manner but is preferably administeredintravenously. Further, the cysteine solution may be diluted (e.g., withsaline, water for injection, and/or an amino acid solution) prior toadministration to the patient.

Other aspects and features of the present disclosure are described inmore detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents an image of vials kept open to air for 25 days. Cysteinewas dissolved at a concentration of 58 mg/mL. The pH of the solution wasnot adjusted (pH 1.3), was adjusted to pH 1.1 with hydrochloric acid, orwas adjusted to pH 1.5 with sodium hydroxide.

FIG. 2A shows an image of vials after being open in the lab for 14 days.Cysteine was dissolved in water (with no pH adjustment) or in acidifiedwater (addition of HCl) at a concentration of 50 mg/ml with stirring for15 or 90 minutes.

FIG. 2B shows an image of vials after being open in the lab for 14 days.Cysteine was dissolved in water (with no pH adjustment) or in acidifiedwater (addition of HCl) at a concentration of 58 mg/ml with stirring for15 or 90 minutes.

DETAILED DESCRIPTION

The present disclosure provides cysteine compositions having low levelsof aluminum. In general, cysteine compositions disclosed herein aresolutions comprising a pharmaceutically acceptable solvent, cysteine,and less than about 400 μg/L of aluminum. In some instances, cysteinesolutions disclosed herein comprise less than about 145 μg/L ofaluminum. Cysteine solutions described herein may be used as additivesto amino acid solutions to meet nutritional requirements of individualsrequiring total parenteral nutrition. Some cysteine solutions disclosedherein may be stable over time (i.e., the level of aluminum isessentially constant and the compositions remain free of visibleparticulate matter). Also provided herein are methods for preparingcysteine compositions, and methods of using cysteine compositions toprovide cysteine to individuals in need thereof.

(I) Cysteine Compositions

One aspect of the present disclosure provides compositions comprisingcysteine. The term “cysteine” as used herein refers to “L-cysteine.” The“L” refers to the orientation of the chiral center of cysteine. Allamino acids in the body are in the “L” configuration. Thus, L-cysteineand cysteine refer to the same moiety and the terms are often usedinterchangeably in clinical and nonclinical literature.

In general, the cysteine compositions disclosed herein are liquidcompositions (e.g., solutions comprising cysteine dissolved in apharmaceutically acceptable solvent). Suitable pharmaceuticallyacceptable solvents include water, acidified water, low concentrationsalt solutions, or saline solutions, which are of sufficient purity tobe included in pharmaceutical formulations. The cysteine solution mayfurther include hydrochloric acid and/or sodium hydroxide (e.g., toadjust the pH).

In general, the cysteine composition or solution has an aluminum contentof less than about 400 μg/L. In certain embodiments, the aluminumcontent may be less than about 350 μg/L, less than about 300 μg/L, lessthan about 200 μg/L, or less than about 150 μg/L. In specificembodiments, the cysteine composition or solution may contain less thanabout 145 μg/L of aluminum. In various embodiments, the aluminum contentmay be less than about 140 μg/L, less than about 135 μg/L, less thanabout 130 μg/L, less than about 125 μg/L, less than about 120 μg/L, lessthan about 115 μg/L, less than about 110 μg/L, less than about 105 μg/L,less than about 100 μg/L, less than 95 μg/L, less than about 90 μg/L,less than about 85 μg/L, less than about 80 μg/L, less than about 75μg/L, less than about 70 μg/L, less than about 65 μg/L, less than about60 μg/L, less than about 55 μg/L, less than about 50 μg/L, less thanabout 45 μg/L, less than about 40 μg/L, less than about 35 μg/L, lessthan about 30 μg/L, less than about 25 μg/L, less than about 20 μg/L,less than about 15 μg/L, less than about 10 μg/L, or less than about 5μg/L. In still other embodiments, the aluminum content may range fromabout 0 μg/L to about 400 μg/L, from about 0 μg/L to about 145 μg/L,from about 0 μg/L to about 100 μg/L, from about 0 μg/L to about 30 μg/L,or from about 0 μg/L to about 10 μg/L.

In general, the cysteine composition or solution is essentially free ofvisible particulate matter. In specific embodiments, the cysteinecomposition or solution is devoid of visible particulate matter.“Visible particulate matter,” as used herein, is defined as precipitatedparticles or crystals, irregularly shaped visible particles or crystals,or crystalline-like visible particles or crystals, but may also includefiber, dust, or other visible particle contaminants. In general, thevisible particulate matter refers to particles or crystals of cystine, adisulfide dimer of cysteine (see Example 2), which forms in the presenceof oxygen (air) and has reduced solubility at higher pH levels. Thecysteine composition or solution may comprise a low percentage ofcystine in solution but be devoid of particles of precipitated cystine.For example, in some embodiments, the cysteine composition or solutionmay comprise less than about 1% by weight of soluble cystine. In certainembodiments, the cysteine composition or solution may comprise less thanabout 0.9 wt %, less than about 0.8 wt %, less than about 0.7 wt %, lessthan about 0.6 wt %, less than about 0.5 wt %, less than about 0.4 wt %,less than about 0.3 wt %, less than about 0.2 wt %, or less than about0.1 wt % of cystine in solution (and be devoid of particles ofprecipitated cystine).

The pH of the cysteine composition or solution may range from about 1.0to about 2.5. In various embodiments, the pH of the cysteine compositionor solution may range from about 1.0 to about 1.25, from about 1.25 toabout 1.5, from about 1.5 to about 1.75, from about 1.75 to about 2.0,from about 2.0 to about 2.25, or from about 2.25 to about 2.5. In someembodiments, the pH of the cysteine composition or solution may be about1.5 or less (e.g., may range from about 1.0 to about 1.5). In otherembodiments, the pH of the cysteine composition or solution may be about1.3 or less (e.g., may range from about 1.0 to about 1.3). In furtherembodiments, the pH of the cysteine composition or solution may be about1.2 or less (e.g., may range from about 1.0 to about 1.2). In additionalembodiments, the pH of the cysteine composition or solution may be about1.1 or less (e.g., may range from about 1.0 to about 1.1). In specificembodiments, the pH of the cysteine composition or solution may be about1.1, about 1.2, or about 1.3.

In general, the cysteine composition or solution may have a dissolvedoxygen content of less than about 8 mg/L. In various embodiments, thedissolved oxygen content of the cysteine composition or solution may beless than about 7 mg/L, less than about 6 mg/L, less than about 5 mg/L,less than about 4 mg/L, less than about 3 mg/L, less than about 2 mg/L,less than about 1 mg/L, less than about 0.8 mg/L, less than about 0.6mg/L, less than about 0.4 mg/L, or less than about 0.2 mg/L. In specificembodiments, the dissolved oxygen content of the cysteine composition orsolution may be less than about 2 mg/L (or 2 ppm).

The cysteine composition or solution may contain a variety of forms ofcysteine. For example, the cysteine composition or solution may containcysteine free base or a pharmaceutically acceptable salt of cysteine.Pharmaceutically acceptable salts of cysteine include, withoutlimitation, acetate, aspartate, benzoate, bitartrate, citrate, formate,gluconate, glucuronate, glutamate, fumarate, hydrochloride,hydrobromide, hydroiodide, hypophosphite, isobutyrate, isocitrate,lactate, malate, maleate, meconate, methylbromide, methanesulfonate,monohydrate, mucate, nitrate, oxalate, phenylpriopionate, phosphate,phthalate, propionate, pyruvate, salicylate, stearate, succinate,sulfate, tannate, tartrate, terephthalate, valerate, and the like. Inexemplary embodiments, the cysteine composition or solution containscysteine hydrochloride, the hydrochloride salt of cysteine. In specificembodiments, the cysteine composition or solution comprises cysteinehydrochloride monohydrate, which is the most soluble form of solidcysteine.

The concentration of cysteine in the cysteine composition or solutionmay vary. For example, the concentration of cysteine may range fromabout 10 mg/mL to about 100 mg/mL. In some embodiments, theconcentration of cysteine in the cysteine composition or solution may berange from about 10-20 mg/mL, from about 20-40 mg/mL, from about 40-60mg/mL, from about 60-80 mg/mL, or from about 80-100 mg/mL. In certainembodiments, the concentration of cysteine may range from about 40 mg/mLto about 60 mg/mL, from about 45 mg/mL to about 55 mg/mL, or from about49 mg/mL to about 51 mg/mL. In specific embodiments, the cysteinecomposition or solution comprises cysteine hydrochloride and theconcentration of cysteine hydrochloride in the composition or solutionmay be about 50 mg/mL.

In general, more than 99% by weight of the total amount of cysteinepresent in the composition or solution is monomeric (and less than 1% byweight of the total amount of cysteine in the composition or solution isa disulfide dimer, i.e., cystine). In various embodiments, more than99.1 wt %, more than 99.2 wt %, more than 99.3 wt %, more than 99.4 wt%, more than 99.5 wt %, more than 99.6 wt %, more than 99.7 wt %, morethan 99.8 wt %, or more than 99.9 wt % of the cysteine is monomeric.

The cysteine compositions or solutions disclosed herein may be dispensedinto glass vials having a thin layer of silica on the interior surface(e.g., silica-lined vials), and wherein the composition or solution isoverlaid with nitrogen prior to sealing of the glass vial. The glassvial may contain 4 mL, 6 mL, 10 mL, 15 mL, 20 mL, 30 mL, 40 mL, or 50 mLof the cysteine composition or solution. In specific embodiments, thevial contains 10 mL of cysteine composition or solution. For example, avial may contain 10 mL of a 50 mg/mL solution of cysteine hydrochloridemonohydrate in water adjusted to pH 1.0 to 2.5. In certain embodiments,more than one vial comprising the cysteine composition or solution ispackaged together for distribution. For example, a package may comprisefive 10-mL vials of cysteine composition or solution as disclosedherein.

The cysteine compositions or solutions, when dispensed into vials asdescribed above, are substantially stable for at least one month, atleast three months, at least six months, at least nine months, at least12 months, at least 18 months, or at least 24 months when stored at 25°C. and 60% relative humidity or 40° C. and 75% relative humidity.Substantially stable means that the level of aluminum does notsubstantially change (e.g., does not increase) from the initial levelpresent when the composition or solution was dispensed into the vial,and the composition or solution remains free of visible particulatematter. In particular embodiments, the composition or solution issubstantially stable i) for at least 18 months when stored at 25° C. and60% relative humidity or ii) for at least 6 months when stored at 40° C.and 75% relative humidity.

The cysteine compositions or solutions provided herein may be formulatedin a variety of dosage forms. Suitable dosage forms include injectableformulations, or oral formulations. In some embodiments, the injectableformulation may be for parenteral administration, e.g., intravenous,intramuscular, intrathecal, and the like. In specific embodiments, theinjectable formulation may be for intravenous administration.

In exemplary embodiments, the cysteine composition is a solutioncomprising 50 mg of cysteine hydrochloride monohydrate dissolved in onemL of water for injection that is adjusted to pH 1.0 to 2.5, and thesolution of cysteine hydrochloride contains less than 145 μg/L ofaluminum. US Pharmacopeia (USP) calls for not less than 85% and no morethan 115% of the labeled amount of cysteine hydrochloride monohydrate.Thus, cysteine compositions labeled with a concentration of 50 mg/mL maycontain from 42.5 mg/mL to 57.5 mg/mL of cysteine hydrochloridemonohydrate.

The 50 mg/mL solution of cysteine hydrochloride monohydrate may have adissolved oxygen content of less than 2 mg/L. The 50 mg/mL solution ofcysteine hydrochloride monohydrate is devoid of visible particulatematter. In some embodiments, 10 mL of the 50 mg/mL cysteinehydrochloride monohydrate is packaged in a silica-lined glass vial. The50 mg/mL solution of cysteine hydrochloride monohydrate is diluted priorto use (e.g., for use as an additive in total parenteral nutritionformulations). In general, the vial comprising the 50 mg/mL solution ofcysteine hydrochloride monohydrate is stored at 20° C. to 25° C. (68° F.to 77° F.). In some embodiments, excursions to 15° C. to 30° C. (59° F.to 86° F.) are permitted [see USP Controlled Room Temperature]. In oneembodiment, the vial is not frozen.

(II) Processes for Preparing Cysteine Compositions

Another aspect of the present disclosure encompasses processes forpreparing the cysteine compositions disclosed herein. In general, theprocesses comprise (a) adding cysteine powder to water to form amixture, (b) mixing the mixture until dissolution is complete, therebyforming a clear solution, (c) filtering the solution to form a filteredsolution, and (d) dispensing the filtered solution into containers orvials, which are then sealed. To minimize oxygen exposure, all media(water, hydrochloric acid (HCl), sodium hydroxide) may be degassed bysparging with nitrogen, and the steps of the process may be conductedunder nitrogen atmosphere (e.g., via use of a mixing bag or atmosbagunder constant flow of nitrogen). All steps of the process generally areperformed at ambient (e.g., room) temperature.

The water used in step (a) may be degassed to the desired level ofdissolved oxygen by sparging with nitrogen. The degassed water may beacidified by adding HCl, wherein the HCl may have been degassed bysparging with nitrogen. The amount of HCl added to the water may vary.In some embodiments, the water may be acidified by adding ⅛^(th) molarequivalent of HCl. In other embodiments, the acidified water may beadjusted to have a concentration of 0.01 N HCl. In additionalembodiments, the acidified water may have a pH of about 1.1. The levelof dissolved oxygen and the pH of the acidified water may be monitoredduring this step of the process. The appropriate mass of cysteine powderis added a volume of acidified water that is close to the final volumeneeded to provide the desired concentration (thus, the concentration ofcysteine at dissolution is nearly the same as the final desiredconcentration of cysteine).

The duration of mixing at step (b) may vary depending upon, for example,the concentration of cysteine, the volume of the mixture, and the like.The duration of mixing is generally just long enough to ensure completedissolution of the cysteine, resulting in formation of a clear solution.Extended mixing times may lead to increased levels of dissolved oxygenin the resulting solution. The mixture may be mixed by stirring with amixing blade or paddle. The rate of stirring can and will vary dependingupon, for example, the concentration of cysteine, the volume of themixture, and so forth. Upon complete dissolution, the pH of the clearsolution may be monitored, and the pH may be adjusted by the addition ofdegassed HCl or degassed NaOH. The volume (or weight) of the clearsolution may be adjusted such that the final concentration of cysteineis at the desired level. For example, additional degassed water may beadded to the clear solution. As mentioned above, the mixing/dissolutionstep may be performed under nitrogen flow.

Step (c) comprises filtering the cysteine solution through at least one0.22 μm filter. The filtration step may reduce the bioburden level inthe cysteine solution and sterilize the solution. The filter maycomprise polyvinylidene difluoride (PVDF), polyethersulfone (PES),cellulose nitrate, cellulose acetate or nylon. In specific embodiments,step (c) comprises serial filtration through two PVDF 0.22 μm filters.The filtration step may be performed under nitrogen flow.

The final step comprises dispensing the filtered cysteine solution intocontainers and sealing the containers. In general, the filtered cysteinesolution is dispensed into silica-lined glass vials. The size orcapacity of the vials may vary, and the volume of filtered cysteinesolution dispensed into a vial may vary. In various embodiments, thevolume of solution dispensed in a vial may be 4 mL, 6 mL, 10 mL, 15 mL,20 mL, 30 mL, 40 mL, or 50 mL. In specific embodiments, vials are filledwith 10 mL of the filtered cysteine solution. The filling may beperformed under nitrogen flow. Once filled with the appropriate volume,the cysteine solution generally is overlaid with nitrogen, and then thevial is sealed using conventional means. For example, the vial may besealed with a cap, e.g., a cap comprising a pierceable septum. Thefilled vials generally are inspected to confirm the correct fill volume,check integrity of the sealing system, and confirm absence ofparticulates (e.g., visible cystine particles, fiber particles, dustparticles). Vials not meeting these criteria are rejected.

The final vials that pass inspection may be packaged in multi-unitpackages. For example, five 10 mL vials may be packaged into onepackage. Different numbers of vials and different sized vials canreadily be packaged into multi-unit packages.

(III) Methods of Using Cysteine Compositions

Still another aspect of the present disclosure encompasses methods ofusing cysteine compositions disclosed herein to provide cysteine toindividuals in need thereof. The disclosed cysteine compositions may beused to meet amino acid nutritional requirements in individualsreceiving total parenteral nutrition. Provided herein are methods forproviding cysteine to individuals in need thereof. In these methods, aninjectable composition that includes cysteine is prepared by admixing acysteine composition described herein with an amino acid injectioncomposition. The injectable composition is then administered to theindividual.

In general, the cysteine compositions disclosed herein can be used tomeet amino acid nutritional requirements in individuals receiving totalparenteral nutrition. In general, total parenteral nutrition tends to beadministered intravenously (e.g., via central venous infusion). Acysteine composition described herein can be added to crystalline aminoacid solution for administration as total parenteral nutrition followingappropriate dilution. The crystalline amino acid solution generallyincludes the nine essential amino acids and may further comprisenonessential amino acids. Total parenteral nutrition can deliver amixture of fluid, electrolytes, sugars, amino acids, vitamins, minerals,and often lipids to individual in need thereof.

Cysteine compositions disclosed herein may be provided to individualswho are unable to receive feedings or fluids by mouth or are unable toabsorb nutrients through the gastrointestinal tract. In specificembodiments, the individual may be a neonate, which is an infant that is28 days of less of age. In other embodiments, the individual may be apreterm infant, who is less than 28 days after term. In still otherembodiments, the cysteine compositions may be administered toindividuals with severe liver disease who may have impairment in theenzymatic conversion of cysteine. In such embodiments, the individualmay be an infant, toddler or child less than 18 years of age.

The dose of cysteine can vary, depending (for example) on the totalamount of amino acids administered to the individual. In general, thetotal amount of amino acids administered to a neonate via a totalparenteral formulation may not exceed 3.5 grams of amino acids per kgper day. In general, the dose of cysteine may range from about 10 mg toabout 50 mg of cysteine per gram of amino acid. In certain embodiments,the dose may be within the range of 15-20 mg of cysteine per gram ofamino acid, the range of about 20-25 mg of cysteine per gram of aminoacid, the range of about 25-30 mg of cysteine per gram of amino acid, orthe range of about 30-40 mg of cysteine per gram of amino acid. Inspecific embodiments, the dose may be about 15 mg, about 22 mg, about 30mg, or about 40 mg of cysteine per gram of amino acid.

In specific embodiments, cysteine compositions disclosed herein may bediluted with a solution of crystalline amino acids prior toadministration to neonates receiving total parenteral nutrition. Forexample, an amino acid admixture may be prepared by adding theappropriate volume of a 50 mg/mL solution of cysteine hydrochloridemonohydrate to a crystalline amino acid solution to provide cysteine at2.2% of the total amino acids being supplied. Thus, a neonate receivingamino acids at 2.5 g/kg/day would be provided 55 mg/kg/day of cysteineor 1.1 mL/kg/day of 50 mg/mL cysteine solution; and a neonate receiving3.0 g/kg/day of amino acids would be provided 1.3 mL/kg/day of 50 mg/mLcysteine solution.

Generally, the injectable composition may be prepared by asepticallydiluting the amino acid admixture with appropriate caloric substrates(e.g., to supply the patient with adequate energy). The admixture may beinspected visually for particulate matter and discoloration prior toadministration. The admixture may be refrigerated until ready for useand typically would be used within 24 hours of mixing. The injectablecomposition comprising cysteine may be administered intravenously. Thoseskilled in the art are familiar with means for administration as well asmeans for determining the rate of administration.

Definitions

When introducing elements of the embodiments described herein, thearticles “a”, “an”, “the” and “said” are intended to mean that there areone or more of the elements. The terms “comprising”, “including” and“having” are intended to be inclusive and mean that there may beadditional elements other than the listed elements.

The term “about,” generally is meant to encompass deviations of plus orminus five percent. However, when used in reference to a labeledpharmaceutical composition, it encompasses deviations of plus or minusfifteen percent (per USP guidelines). For example, a cysteinecomposition labeled with a concentration of 50 mg/mL may contain from42.5 mg/mL to 57.5 mg/mL of cysteine hydrochloride monohydrate.

EXAMPLES

The following examples illustrate various embodiments of the presentdisclosure.

Example 1: Reduction in Aluminum Levels

Solutions of cysteine hydrochloride tend to be acidic (e.g., have a pHfrom −1.0 to 2.5), suggesting that they could leach aluminum fromstandard borosilicate glass vials. To address this issue, several vialtypes were surveyed in a study (summarized in Table 1). In this study,aluminum content in 50 mg/mL solution of cysteine HCl was comparedacross various vials that were or were not subjected to terminalsterilization via heating. The study also evaluated aluminum content inthe unapproved reference drug. The study showed that the use of Type I+vials, which have an interior silica lining, greatly limited thealuminum content. In the study, aluminum contents in Type I+ vials wereobserved to be <10 μg/L for all samples and conditions tested, which is2-3 orders of magnitude lower than observed for the reference drugproduct and for other unlined vial types.

TABLE 1 Summary of aluminum content results in different vial typessubjected to different terminal sterilization conditions Time AluminumVial Treated at Content, Sample size Vial Type 121° C. μg/L Bulk AV- NANA None 6.2 FFP-17- 0006 AV-FFP- 10-mL Type 1+, lined None 4.2 17-000610-mL Type 1, unlined, molded None 33.9 50-mL Type 1, unlined, molded ANone 17.4 50-mL Type 1, unlined, molded B None 26.3 10-mL Type 1+, lined20 min 7.3 10-mL Type 1, unlined, molded 20 min 60.9 50-mL Type 1,unlined, molded A 20 min 52.5 50-mL Type 1, unlined, molded B 20 min91.3 Reference 10-mL Unknown None 427 drug 10-mL Unknown 20 min 560

Example 2: Identification of Particulate Matter as Cystine

During scale-up production of cysteine hydrochloride, about 40-60% theAPI-filled vials were rejected during visual inspection because solidparticles appeared after the vials were stored for several days at roomtemperature. The colorless, flat, irregularly shaped,crystalline-appearing particles were identified as cystine by acombination of Fourier Transform Infrared Spectroscopy (FTIR) andstereomicroscopy techniques.

Example 3: Solubility of Cysteine and Cystine

Cystine, a sulfur-sulfur dimer of cysteine, can form in the presence ofoxygen (air) in cysteine solutions. Cystine, especially the free base,is several orders of magnitude less soluble in aqueous solutions thancysteine, and the solubility of cystine decreases as the pH increases.Thus, if enough cystine dimer is formed in the cysteine hydrochlorideInjection drug product, the cystine may precipitate, or form undesirableparticulates, in the solution. A summary of the solubility of thecystine and cysteine species is shown in Table 2.

TABLE 2 Summary of solubility results for cysteine and cystine speciesSolubility (mg/mL) Species Media at 22° C. Cystine free base Water 0.14Cysteine HCl Injection, 0.59 50 mg/mL Cystine dihydrochloride Water 1.04Cysteine HCl Injection, 9.93 50 mg/mL Cysteine free base Water 18.74Cysteine hydrochloride Water 493.99

Furthermore, the solubility of cystine increases as the pH decreases.For example, at pH 1.4, the solubility of cystine is approximately 1mg/ml, whereas at pH 1.0, its solubility increases to approximately 4mg/mL (Carta et al., J Chem Eng Data, 1996, 41(3):414-417).

Example 4: Solubilization at 50 mg/mL at pH 1.1 or pH 1.3

To determine whether the formation of cystine particulates could bereduced, the process for preparing cysteine hydrochloride injection wasmodified by degassing solutions to reduce the levels of oxygen andadjusting the pH to a lower level. In particular, water was degassed bynitrogen sparging to reach various levels of dissolved oxygen (DO): 0.2mg/L, 2.6 mg/L, and 7.0 mg/L. The pH of the final solution was 1.3-1.4.One part of the solution was adjusted to pH 1.1 by addition of 5N HCl(which had been degassed by nitrogen sparging). The water (at thedifferent pH levels) was added to cysteine hydrochloride monohydratepowder at 50 mg/mL (under N2 flow). The solution was stirred untildissolution was complete and the solution was clear. The solution wasfiltered, dispensed into (type 1+ silica lined) vials, and sealed undernitrogen. In order to have a reference at a saturated oxygen value, onevial of each series was exposed to air for several seconds beforesealing. The protocol is outlined below in Table 3.

TABLE 3 Solubilization at 50 mg/mL Opened in the lab pH adjustment %DO/DO mg/L before sealing No pH adjustment (~1.3)  2.6%/0.22 mg/L No NoYes Kept open in the lab 29.4%/2.55 mg/L No No Yes Aerated water No81.0%/7.02 mg/L No pH adjustment to ~1.1  2.6%/0.22 mg/L No No Yes29.4%/2.55 mg/L No No Yes

No particles were visible after 40 days in the vials sealed undernitrogen whatever the level of dissolved oxygen (DO) and whatever thepH. Particle formation was only observed for the vial left open in thelab. In increase in the level of dissolved oxygen is expected when vialsare exposed to air.

Example 5: Solubilization at 58 mg/mL at pH 1.1, pH 1.3, or pH 1.5

Generally, cysteine HCl is prepared at an intermediate concentration(˜58 mg/mL) and then adjusted to 50 mg/mL prior to filtration andbottling. Cysteine hydrochloride monohydrate powder was added undernitrogen flow to water degassed by nitrogen sparging (to reach 0.2 mg/Lor 2.4 mg/L of dissolved oxygen). The pH was about 1.3 in the finalsolution. In one fraction, the pH of the solution was adjusted to pH 1.1by addition of 5N HCl (previously degassed by nitrogen sparging). Inanother fraction, the pH was adjusted to 1.5 with 0.1 N NOH (previouslydegassed). The solutions were stirred until dissolution was complete,diluted to 50 mg/ml with water (previously degassed), filtered, andfilled into (lined) vials, which were then sealed under nitrogen. Inorder to have a reference at a saturated oxygen value, one vial of eachseries was exposed to air, and one vial of each series was kept open inthe lab.

TABLE 4 Solubilization at 58 mg/mL Opened in the lab pH adjustment %DO/DO mg/L before sealing No pH adjustment (~1.3)  2.4%/0.22 mg/L Yes NoNo Kept open in the lab 27.3%/2.43 mg/L Yes No No Kept open in the labpH adjusted to ~1.1  2.4%/0.22 mg/L Yes No No Kept open in the lab27.3%/2.43 mg/L Yes No No Kept open in the lab pH adjusted to ~1.3 2.4%/0.22 mg/L Yes No No Kept open in the lab 27.3%/2.43 mg/L Yes No NoKept open in the lab

No particles were visible (observation over 80 days) in the vials sealedunder nitrogen whatever the level of dissolved oxygen (DO) and whateverthe pH. In contrast, many particles appeared in samples exposed or keptopen in air at pH 1.3 or 1.5 after 5 to 13 days (FIG. 1). A fewparticles were also observed after 25 days at pH 1.1. HCl (5M) was addedto the pH 1.3 samples exposed to air (many particles) to decrease the pHto 1.1. A complete dissolution of the particles was observed after 8 hrwithout stirring, presumably due to re-solubilization of cystine at thelower pH.

The following conclusion can be drawn from the data of Examples 4 and 5:(i) air exposure and oxidation foster particle formation, (ii) acidic pHappears to slow down oxidation and cystine precipitation, and (iii) anintermediate concentration of 58 mg/mL appears to promote particleformation.

Example 6: Comparative Study

A study was undertaken to directly compare (i) 50 mg/mL vs. 58 mg/mLdissolution concentration of cysteine HCl, (ii) dissolution in water vs.dissolution in acidic water (addition of 5N HCl to obtain a finalconcentration of 0.01 N HCl), and (iii) 15 min vs. 90 min duration ofstirring during dissolution. All experiments were carried out inparallel and launched the same day using Type 1+ silica-lined vials. Allsteps were performed under nitrogen with low dissolved oxygen levels(less than 0.5 mg/L). For each series, 3 vials were sealed undernitrogen and one vial was exposed to air to assess accelerated oxidizingconditions.

No particles were observed after 66 days under any condition for thevials sealed under nitrogen. In vials left open, particles were observedafter 7 days in those with no pH adjust and high dissolutionconcentration (58 mg/mL). FIGS. 2A and 2B presents images at 14 days invials kept open in the lab. For these samples, the duration of stirringincreased the abundance of particles in vials with higher pH (i.e.,those without pH adjustment) (FIG. 2B). On the contrary, decreasing thepH to about 1.1 helped prevent formation of particles whatever thestirring duration (FIG. 2A).

In samples without pH adjustment and exposed to air for longer than 14days, particles were observed after 37 days in those dissolved at 50mg/mL and stirred for 90 min. Particles were observed after 66 days inthose dissolved at 50 mg/mL and stirred for 15 min. In contrast, noparticles were observed after 66 days in samples with a more acidic pH(pH 1.1), regardless of dissolution concentration and stirring duration.

From the studies described above, the manufacturing process was modifiedto ensure that all steps were conducted in low oxygen environment, allliquids were degassed to reduce dissolved oxygen levels, and the initialwater used to dissolve the cysteine was acidified. Adopting thesechanges reduced the percentage of rejected vials (e.g., containingparticulates) per batch as high as about 50% to less than about 0.05%.

Example 7: Stability Studies

Vials of 50 mg/mL of cysteine HCl from various batches were stored atroom temperature and assayed at selected time points. The data arepresented below in Table 5. (1 ppb=1 μg/L)

TABLE 5 Stability at room temperature Sample Time Cysteine CystineAluminum Lab Batch, lined vial 18 mo 100.8%  1.2%  <3 ppb Batch 1700141,unlined vial 12 mo  99.7% 1.05% 182 ppb 18 mo  97.5% 1.23% 370 ppb Batch1700196, unlined vial  6 mo  98.4% 0.84% 189 ppb 12 mo  98.2% 1.17% 273ppb Batch 1800029, lined vial  6 mo  98.5% 0.83%  <3 ppb  9 mo  97.8%0.91%  <3 ppb Batch 1800030, lined vial  6 mo  98.6% 0.91%  <3 ppb  9 mo 96.7% 0.98%  <3 ppb Batch 1800031, lined vial  6 mo  98.2% 1.00%  <3ppb  9 mo  95.6% 1.10%  <3 ppb Lab batch, low pH, lined vial  3 mo101.2% 0.19%  <3 ppb Batch 1800172, lined vial  0 mo  99.2% 0.46%  <3ppb Batch 1800229, lined vial  0 mo  98.1% 0.66%  <3 ppb

What is claimed is:
 1. A cysteine hydrochloride solution comprisingabout 40 mg/mL to about 60 mg/mL of cysteine hydrochloride and a pH fromabout 1.0 to about 1.5, wherein preparation of the cysteinehydrochloride solution comprises: acidifying water to a pH from about1.0 to about 1.5 to form acidified water; dissolving about 40 mg toabout 60 mg of cysteine hydrochloride per 1 mL of acidified water toform a solution; and filling and sealing a container in which thesolution is disposed under flow of an inert gas.
 2. The cysteinehydrochloride solution of claim 1, which is free of visible particulatematter.
 3. The cysteine hydrochloride solution of claim 1, which is freeof visible particulate matter when stored for at least six months at 25°C. and 60% relative humidity.
 4. The cysteine hydrochloride solution ofclaim 1, which is free of visible particulate matter when stored for atleast 12 months at 25° C. and 60% relative humidity.
 5. The cysteinehydrochloride solution of claim 1, which is free of visible particulatematter when stored for at least 18 months at 25° C. and 60% relativehumidity.
 6. The cysteine hydrochloride solution of claim 1, which isfree of visible particulate matter when stored for at least 24 months at25° C. and 60% relative humidity.
 7. The cysteine hydrochloride solutionof claim 1, which has a cystine content of less than about 1% by weightof the total cysteine present in the cysteine hydrochloride solution. 8.The cysteine hydrochloride solution of claim 1, which has a dissolvedoxygen content of less than 2 mg/L.
 9. The cysteine hydrochloridesolution of claim 1, wherein the pH of the cysteine hydrochloridesolution is from about 1.0 to about 1.4.
 10. The cysteine hydrochloridesolution of claim 1, wherein the pH of the cysteine hydrochloridesolution is from about 1.0 to about 1.3.
 11. The cysteine hydrochloridesolution of claim 1, wherein the pH of the cysteine hydrochloridesolution is from about 1.0 to about 1.2.
 12. The cysteine hydrochloridesolution of claim 1, having an aluminum content of less than about 200μg/L.
 13. The cysteine hydrochloride solution of claim 1, having analuminum content of less than about 145 μg/L.
 14. The cysteinehydrochloride solution of claim 1, having an aluminum content of lessthan about 100 μg/L.
 15. The cysteine hydrochloride solution of claim 1,having an aluminum content of less than about 30 μg/L.
 16. The cysteinehydrochloride solution of claim 1, having an aluminum content of lessthan about 10 μg/L.
 17. The cysteine hydrochloride solution of claim 1,having an aluminum content from about 0 μg/L to about 100 μg/L.
 18. Thecysteine hydrochloride solution of claim 1, having an aluminum contentfrom about 0 μg/L to about 30 μg/L.
 19. The cysteine hydrochloridesolution of claim 1, having an aluminum content of less than about 145μg/L when stored for at least six months at 25° C. and 60% relativehumidity.
 20. The cysteine hydrochloride solution of claim 1, having analuminum content of less than about 145 μg/L when stored for at least 12months at 25° C. and 60% relative humidity.
 21. The cysteinehydrochloride solution of claim 1, having an aluminum content of lessthan about 145 μg/L when stored for at least 18 months at 25° C. and 60%relative humidity.
 22. The cysteine hydrochloride solution of claim 1,having an aluminum content of less than about 145 μg/L when stored forat least 24 months at 25° C. and 60% relative humidity.
 23. The cysteinehydrochloride solution of claim 1, which comprises about 50 mg/mL ofcysteine hydrochloride.
 24. The cysteine hydrochloride solution of claim1, wherein the acidifying is performed under flow of an inert gas. 25.The cysteine hydrochloride solution of claim 1, wherein the preparationof the cysteine hydrochloride solution further comprises sparging waterwith an inert gas to achieve a dissolved oxygen content of less than 2mg/L in the water prior to the acidifying.
 26. The cysteinehydrochloride solution of claim 1, wherein the dissolving comprisesstirring under flow of an inert gas.
 27. The cysteine hydrochloridesolution of claim 1, wherein the preparation of the cysteinehydrochloride solution further comprises sterile filtering the solutionprior to the filling and sealing.
 28. The cysteine hydrochloridesolution of claim 27, wherein the sterile filtering comprises passingthe solution through at least one 0.22 μm filter.
 29. The cysteinehydrochloride solution of claim 1, wherein the acidifying, thedissolving, and the filling and sealing are performed at ambienttemperature.
 30. The cysteine hydrochloride solution of claim 1, whereinthe container is a silica-lined glass vial.